Bacteriophage-mediated immunisation

ABSTRACT

The present invention relates to vaccines comprising a bacteriophage which has been engineered to express an immunogenic protein/peptide and wherein the surface of the bacteriophage has not been modified to contain proteins/peptides designed to target the phage to receptors on the surface of specific cell types.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 371 from PCTApplication No. PCT/GB02/01413, filed in English on Mar. 25, 2002, whichclaims the benefit of Great Britain Application Serial No. 0107319.6filed on Mar. 23, 2001, the disclosures and contents of which areincorporated by reference herein in their entireties.

The present invention relates to vaccines comprising a bacteriophageengineered to express an immunogenic protein/peptide.

Genetic vaccination is a new and exciting technology in which nucleicacid is used as the vaccine material (for review see Leitner et al.2000. Vaccine 18: 765–777). In contrast, traditional vaccines requirethe use of pathogenic microbes or their antigenic components. There arethree classes of “traditional” vaccines: attenuated, killed/subunit, andrecombinant. Attenuated vaccines are live microorganisms with reducedpathogenicity and are generally the most effective vaccines. However,they can produce complications if the vaccine agent grows unchecked orreverts to a more pathogenic form. Killed or subunit vaccines requiremultiple injections, thereby increasing cost and creating logisticalproblems, and may contain incompletely killed microbes. Recombinantvaccines, in which an antigen from a pathogenic organism is engineeredinto a non-pathogenic vector can be effective, but difficulties inachieving expression of the antigen in a native conformation often limitefficacy.

To be effective, vaccines need to provide a sufficient dose of antigenfor time periods long enough to induce a secondary (memory) response.This poses a problem for traditional vaccines; DNA/RNA vaccines,however, can effectively produce copies of pathogenic antigens for longperiods of time, and thus induce both MHC Class I & II responses, asseen with live vaccines. However, for all their promise, DNA vaccineshave yet to fulfil their full potential. Despite eliciting a measurablehumoral (antibody) immune response, many DNA vaccines exhibit poorefficacy when challenged with the infective organism (Beard, C W &Mason, P W. 1998. Nature Biotech. 16: 1325).

The mechanism by which the nucleic acid enters host cells and induces animmune response is unclear at present. The simplest technique is toadminister the DNA as a soluble injection, usually givenintramuscularly. Two other techniques in common use are “gene gun”technology, in which DNA is precipitated onto tiny gold particles whichare forced into cells with a helium blast, or liposome-mediatedtransfection, in which DNA is coated with positively charged lipid toform a complex which fuses with the host cell membrane. It is believedthat cells surrounding the immunisation site take up the DNA, expressthe encoded antigen(s), and are recognised as “foreign” by antigenpresenting (AP) cells of the immune system, which then proceed toactivate T and B cells to elicit an immune response against the antigen.

Limitations would appear to be: (1) Expression is relatively inefficientand non-specific, with the majority of the DNA being expressed in non-APcells; (2) Expression of foreign antigens in non AP-cells willeventually lead to the death of that cell due to its recognition asbeing “infected” by the host immune system, thus shortening thepotential immune response; and (3) Naked DNA/RNA is highly sensitive tothe action of nucleases. It is likely that the majority of nucleic acidused for immunisation is degraded shortly following immunisation.

WO98/05344 describes a method for delivering exogenous genes using abacteriophage vector wherein the bacteriophage vector has been modifiedto include on its surface a ligand that binds to a receptor on a targetcell. The vectors described are generally intended to be used for genetherapy applications where the vectors are targeted to specific celltypes. There is also mention of using the modified bacteriophage vectorsto deliver antigenic peptides.

U.S. Pat. No. 5,736,388 describes modified lamboid bacteriophage fordelivering nucleic acid molecules to eukaryotic cells in which thebacteriophage has been modified by incorporating mutant tail fibreproteins or by incorporating ligands for eukaryotic cell receptors.

U.S. Pat. No. 6,054,312 relates to filamentous phage particlesdisplaying a ligand on their surface, the ligand being a fusion proteinwith a phage capsid protein, covalently conjugated to phage particles,or complexed with modified phage particles.

WO99/55720 also describes phage which have been modified to externallydisplay a heterologous targeting protein for use in targeted genedelivery.

However, the aforementioned patents/patent applications all describemodifying the surface of the phage so as allow targeted delivery ofnucleic acid to specific cells, generally for gene therapy purposes.

A number of documents (Ishiura, M. et al, Molec. And Cell. Biol.,p607–616, 1982; Aujame, L. et al, Biotechiques, 28 p1202–1213, 2000;Horst, J. et al, Proc. Natl. Acad. Sci., 72, p3531–3535, 1975; Jkayamaand Dery, Molec. and Cell. Biol. 5, p1136–1142, 1985; and Srivatsan, E.et al, 38, p227–234, 1984) relate to the use of phage to transfectcultured mammalian cells and express protein therein. However, there isno suggestion that this could be applied in vivo, or used in thedevelopment of vaccines.

It is an object of the present invention to obviate and/or mitigate atleast one of the aforementioned disadvantages.

In a first aspect the present invention provides a vaccine formulationcomprising a bacteriophage particle the surface of which is unmodifiedand a pharmaceutically acceptable carrier therefor, the bacteriophageparticle comprising an exogenous nucleic acid molecule encoding apolypeptide which is capable of expression and presentation on thesurface of an antigen presenting cell of an organism, such that animmune response to said polypeptide is raised in the organism.

Unlike previous disclosures, see for example WO98/05344, U.S. Pat. No.5,736,388, U.S. Pat. No. 6,054,312 and WO99/55720, it is to beappreciated that the surface bacteriophage of the present invention hasnot been modified to comprise exogenous peptides/proteins (ie.peptides/proteins not normally present) on the surface of the phage,designed to target the phage to receptors on the surface of specificcell types. It is to be understood therefore that the surface of thebacteriophage may be modified to comprise exogenous peptides/proteinsnot designed to target the phage to receptors on the surface of specificcell types.

The present inventors have observed that bacteriophage which have notbeen modified to comprise targeting peptides or ligands on the surfaceof the bacteriophage particle are taken up by AP cells. Thus, thebacteriophage of the present invention are thought to be recognised as“foreign” and are therefore processed in the normal manner by a host'simmune system. Moreover, by modifying the genome of the bacteriophage toinclude exogenous nucleic acid capable of encoding a foreignpeptide/protein, that is a peptide/protein not normally present in achosen mammalian host, an immune response to this foreign protein iselicited. Thus, the nucleic acid encoding the foreign peptide/protein isexpressed (in an antigen present cell or otherwise) and presented on thesurface of the AP cell. It is to be appreciated that the immune responsemay be a humoral (ie. antibody) and/or cellular immune response.

Exogenous nucleic acid relates to a non-naturally occurringpolynucleotide that is capable of being expressed as an heterologouspeptide or protein, that is a peptide or protein which is not normallyexpressed or is expressed at biologically insignificant levels in anaturally-occurring bacteriophage. The expressed peptide or protein isexpressed at a level sufficient to elicit an immune response in a hostto which the vaccine has been presented.

It is to be appreciated that the present invention is applicable to thepreparation of a vaccine for practically any infectious disease,providing that a suitable immuno-protective response can be raised to aprotein or proteins of an infectious agent. Examples of suitablediseases include vaccination directly against the disease-causing agent,or alternatively, vaccination against the disease-carrying vector. Suchinfectious agents or vectors include virus, bacteria, fungi, yeast,protozoa, helminths, insecta, and transmissible spongiformencephalopathies. The present invention would be applicable toinfectious diseases of both humans and animals. Lists of suitablediseases are well known to those versed in the art and examples are tobe found in the O.I.E. Manual of Standards and Diagnostic Tests 3rd Ed.,OIE, Paris 1996, Topley & Wilson's Principles of Bacteriology, Virologyand Immunity 8th Ed., Eds. Parker M. T. and Collier L. H., Vol IV(Index), Edward Arnold, London 1990, The Zoonoses: InfectionsTransmitted from Animals to Man. Bell J. C. et al., Edward Arnold,London 1988 and Parasitology: The Biology of Animal Parasites 6th Ed.Noble E. R. et al., Lea & Febiger, Philadelphia, 1989. In addition thepresent invention could be used to elicit an immune response againstcancer cells by means of the expression of a cancer cell specificantigen as the vaccine protein.

The present invention thus provides a way of encapsulating exogenousnucleic acid eg. DNA inside a stable matrix, in order to protect it fromfor example nucleases present for example in cells. The “foreign”proteins on the surface of the bacteriophage allow direct uptake ofnucleic acid specifically to antigen presenting (AP) cells. Withoutbeing bound by theory it is expected the bacteriophage particle isrecognised as a foreign antigen. The entire particle is thus taken updirectly by the antigen presenting cells of the host immune system,where the protein coat is removed, releasing the DNA which may then moveinto the nucleus and be expressed. This procedure, is thought to beefficient, since vaccine DNA expression and subsequent polypeptideproduction should only take place in AP cells; the optimum route forinducing an immune response.

In general the term “polypeptide” refers to a chain or sequence of aminoacids displaying an antigenic activity and does not refer to a specificlength of the product as such. The polypeptide if required, can bemodified in vivo and/or in vitro, for example by glycosylation,amidation, carboxylation, phosphorylation and/or post translationalcleavage, thus inter alia, peptides, oligo-peptides, proteins and fusionproteins are encompassed thereby. Naturally the skilled addressee willappreciate that a modified polypeptide should retain physiologicalfunction i.e. be capable of eliciting an immune response.

The bacteriophage of the present invention preferably containappropriate transcription/translation regulators such as promoters,enhancers, terminators and/or the like. Typically the promoter may be aeukaryotic promoter such as CMV, SV40, thymidine kinase, RSV promoter orthe like. Conveniently the promoter may be a constitutive promoter.However, controllable promoters known to those of skill in the art mayalso be used. For example constructs may be designed which comprise theexogenous nucleic acid under control of a constitutive promoter and acontrollable promoter. In this manner it may be possible to causeexpression of the exogenous nucleic acid initially by way of theconstitutive promoter and at a second time point by expression from thecontrollable promoter. This may result in a stronger immune response.

Many suitable bacteriophage are known to those skilled in the art. Anexample of a suitable bacteriophage is lambda (λ). Currently,bacteriophage λ is used as a cloning vector during routine DNAmanipulation procedures. For these, the DNA is purified away from thephage structure. However, an intact λ phage particle fulfils thecriteria listed above; the DNA is contained within a protective proteinmatrix which is recognised as a foreign antigen by the host immunesystem. Phage λ normally infects the bacterium E. coli, and its DNA isthought to be “inert” in a eukaryotic cell (ie. it will not beexpressed). However, if a eukaryotic promoter is incorporated upstreamof the vaccine (or foreign) gene of interest, then expression of thatgene to provide an antigen ie. protein/peptide should occur if the DNAis taken up by a mammalian cell. Due to extensive use as a routinecloning vector, many variants of λ exist, including some with strongeukaryotic promoters designed to direct expression in mammalian cells.Normally, the relevant section of the λ vector is removed as plasmid DNAprior to further genetic manipulations:—highly purified plasmid DNA froman E. coli host will then be used for genetic immunisation. However, ifan intact λ phage particle containing a eukaryotic promoter and thevaccine (ie. exogenous) gene of interest is used for immunisation, it istaken up by AP cells. Following protein coat removal, antigen productiondirectly within the AP cell is thought to occur and antigen presented onthe surface of the AP cells so as to induce an immune response. In thiscase only the most basic purification procedure is required to producephage particles ready for immunisation. An additional advantage of usingλ compared to plasmid cloning vectors is that much larger insert sizescan be accommodated.

Other suitable bacteriophage are well known to those of skill in the artand include p1 phage, T phages (eg. T1–7), Mu, fd or M13, as well asfilamentous phage.

Preferred bacteriophage of the present invention have the ability toincorporate exogenous nucleic acid and associated promoters, enhancers,terminators and/or the like of between about 0.5–100 kilobases. Forexample known lambda phages can accommodate between 9–50 kilobases. Inthis manner it is possible to express single or multiple copies of apeptide/protein or a plurality of peptides/proteins.

Typically, the bacteriophage of the present invention are abortive tolytic growth in the natural bacterial flora of the chosen mammalianhost. Many “laboratory” strains of phage are known for example which areonly able to infect non-wild type “laboratory” bacterial strains.Additionally or alternatively the bacteriophage may be abortive to lyticgrowth of the host bacterial strain in vitro, or require helper phage togrow in vitro. Thus the bacteriophage may contain for example an ambermutation, a temperature sensitive mutation or the like.

Means are generally provided to enhance expression of the exogenousnucleic acid in the AP cells. Such means include methods to helpminimise nucleic acid degradation and/or targeting to the nucleus.Examples of such means include the use of chloroquine or otherinhibitors of lysosomal/endosomal enzymic catabolism to minimise nucleicacid degradation and/or the use of nuclear localisation signals todirect the nucleic acid to the nucleus.

The vaccine formulation may further comprise a source of the proteinwhich is to be expressed by the bacteriophage. In this manner a host mayelicit a primary immune response to the protein and thereafter elicit afurther or sustained immune response due to the protein being expressedand presented on the surface of an AP cell.

In a further embodiment the phage could be modified to also express theantigenic protein on the surface of the phage particle. For example itis possible to use intact bacteriophage M13 particles as a vectorvehicle. Insert sizes for M13 are considerably smaller than for λ, butthe use of “Phage Display” technology (Hawkins, R E et al. 1992, J. Mol.Biol. 226: 889) means that the phage particle can carry a portion offoreign antigen fused to its coat protein. Thus a construct can be madein which the vaccine gene is under control of both a prokaryotic (eg.Lac Z promoter) and a eukaryotic promoter (eg. CMV promoter): when grownin an E. coli host, the prokaryotic promoter will direct expression ofthe vaccine antigen and allow its incorporation into the M13 coat as aprotein conjugate, which should elicit a strong primary responsefollowing vaccination. Thereafter, following uptake by AP cells, the DNAwill be released and the eukaryotic promoter will direct long-lastingexpression of the vaccine antigen from within the AP cell, maintaining astrong secondary response.

The exogenous nucleic acid may encode at least a further polypeptide(s),such as a polypeptide capable of augmenting the immune response. Thefurther polypeptide may be an adjuvant protein or polypeptide, such as acytokine coding, for example, for an interferon such as γ interferon(γIFN), IL-2, IL-6, IL-7, IL-12, CM-CSF and/or othercytokines/chemokines. Moreover, “helper epitopes”, such as HepB coreantigen may be used to activate B cells and elicit strong T-cellresponses. Alternatively or additionally, immunostimulatory signals suchas CpG oligodinucleotides may be used.

The bacteriophage may be administered by any suitable route, for exampleby injection and may be prepared in unit dosage in for example ampules,or in multidose containers. The bacteriophage may be present in suchforms as suspensions, solutions, or emulsions in oily or preferablyaqueous carriers. Alternatively, the bacteriophage may be in lyophilizedform for reconstitution, at the time of delivery, with a suitablecarrier, such as sterile pyrogen-free water. In this manner stabilisingagents, such as proteins, sugars etc. may be added when lyophilising thephage particles. Both liquid as well as lyophilized forms that are to bereconstituted will comprise agents, preferably buffers, in amountsnecessary to suitably adjust the pH of the injected solution. For anyparenteral use, particularly if the formulation is to be administeredintravenously, the total concentration of solutes should be controlledto make the preparation isotonic, hypotonic, or weakly hypertonic.Nonionic materials, such as sugars, are preferred for adjustingtonicity, and sucrose is particularly preferred. Any of these forms mayfurther comprise suitable formulatory agents, such as starch or sugar,glycerol or saline. The compositions per unit dosage, whether liquid orsolid, may contain from 0.1% to 99% of bacteriophage material.

In a preferred presentation, the vaccine can also comprise an adjuvant.Adjuvants in general comprise substances that boost the immune responseof the host in a non-specific manner. A number of different adjuvantsare known in the art. Examples of adjuvants may include Freund'sComplete adjuvant, Freund's Incomplete adjuvant, liposomes, and niosomesas described, for example, in WO 90/11092, mineral and non-mineraloil-based water-in-oil emulsion adjuvants, cytokines, shortimmunostimulatory polynucleotide sequences, for example in plasmid DNAcontaining CpG dinucleotides such as those described by Sato Y. et al.(1996) Science Vol. 273 pp. 352–354; Krieg A. M. (1996) Trends inMicrobiol. 4 pp. 73–77.

The bacteriophage may also be associated with a so-called “vehicle”. Avehicle is a compound, or substrate to which the bacteriophage canadhere, without being covalently bound thereto. Typical “vehicle”compounds include gold particles, silica particles such as glass and thelike. Thus the bacteriophage of the invention may be introduced into anorganism using biolistic methods such as the high-velocity bombardmentmethod using coated gold particles as described in the art (Williams R.S. et al. (1991) Proc. Natl. Acad. Sci. USA 88 pp. 2726–2730; Fynan E.F. et al. (1993) Proc. Natl. Acad. Sci. USA Vol. 90 pp. 11478–11482).

In addition, the vaccine may comprise one or more suitablesurface-active compounds or emulsifiers, eg. Span or Tween.

The mode of administration of the vaccine of the invention may be by anysuitable route which delivers an immunoprotective amount of the virus ofthe invention to the subject. However, the vaccine is preferablyadministered parenterally via the intramuscular or deep subcutaneousroutes. Other modes of administration may also be employed, wheredesired, such as via mucosal routes (eg. rectal, oral, nasal or vaginaladministration) or via other parenteral routes, ie., intradermally,intranasally, or intravenously. Formulations for nasal administrationmay be developed and may comprise for example chitosan as an adjuvant(Nat. Medicine 5(4) 387–92, 1999).

It will be understood, however, that the specific dose level for anyparticular recipient organism will depend upon a variety of factorsincluding age, general health, and sex; the time of administration; theroute of administration; synergistic effects with any other drugs beingadministered; and the degree of protection being sought. Of course, theadministration can be repeated at suitable intervals if necessary.

In a further aspect therefore, the present invention provides a methodof immunising, prophylactically and/or therapeutically, a human oranimal, comprising administering to the human and/or animal an effectivedose of a vaccine formulation as described herein. It being understoodthat an effective dose is one which is capable of eliciting an immuneresponse in the human and/or animal.

The present invention will now be further described by way of Exampleand with reference to the following Figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the antibody responses in mice vaccinated with thebacteriophage based vaccine of the present invention, as compared toconventional intro-muscular DNA vaccination and a control;

FIG. 2 shows the relative amounts of IgG and IgM produced by thevaccinated mice;

FIG. 3 shows the uptake and expression of foreign DNA by macrophages invitro;

FIG. 4 shows antibody responses against whole MmmSC antigen in micevaccinate with λ-MmmSC library. Total antibody responses against MmmSCsonicated whole cell extracts from mice vaccinated with lambda MmmSCexpression library (λMmmSC). Responses were measured by ELISA. Theresponses from four mice vaccinated with λMmmSC are shown by unbrokenlines. A representative response from a control mouse is shown by adotted line. Times of vaccination are shown by the arrows (day 0 and day28);

FIG. 5 shows (A) Immunoblot of purified GFP probed with final bleedsfrom mice immunised with λ-GFP (left; 8 lanes, 1 per mouse), plasmidpEGFP-C1 (middle; 6 lanes, 1 per mouse), and positive control (rabbitanti-GFP). The remaining two mice vaccinated with pEGFP-C1 were testedon a different immunoblot and did not reveal a response against pureGFP. (B) Immunoblot against λ proteins with time course sera from 3representative animals vaccinated with λ-GFP (left) or pEGFP-C1 (right);

FIG. 6 shows mouse peritoneal macrophages incubated with DNA vaccinesand probed with specific antisera: A, ×10 magnification; B, 40×magnification: i) no vaccine. Probed with anti-GFP; ii) incubated withλB1 (MmmSC pdh gene) and probed with anti-MmmSC IgG; iii) incubated withλ-GFP and probed with anti-GFP; and iv) incubated with plasmid pEGFP andprobed with anti-EGFP; AND

FIG. 7 ELISA measure of anti-GFP signal from macrophages incubated withλEGFP, pEGFP-Cl or a non-expressing λ control (λcI⁸⁵⁷). Three separateassays were run for macrophages incubated with λEGFP (1) or pEGFP-Cl (2)and the mean values and standard deviations of the anti-GFP signals areshown. Two separate assays were run for the negative controls, these areplotted individually. Assay (3) shows the secondary antibody only signalagainst λEGFP, which assay (4) shows the non-specific signal from bothprimary and secondary antibodies against bacteriophage λ proteins.

EXAMPLE 1

A phage expression library was constructed by cloning semi-randomfragments (Tsp509I: cuts at AATT) of the genome of Mycoplasma capricolumsubspecies capripneumoniae (Mccp) strain F38 into the EcoRI site of thelambda ZAP express expression vector (made by Stratagene). This phagecontains a cytomegalovirus promoter sequence, and a LacZ promoter soinserts can be expressed in both prokaryotic and eukaryotic cells.

The test “library” used, based upon mycoplasmal DNA is likely to givepoorer results compared to more suitable constructs, and resultsobtained should be interpreted in the light of this. It is very likelythat a more suitable library construct would gave vastly betterresponses. Mycoplasmal DNA is very AT rich (Mccp DNA is 75% AT). Thusaberrant expression from AT-rich promoter-like sequences is likely tooccur at a high frequency. In addition, codon usage of mycoplasmal DNAis unusual; the codon for tryptophan is UGA, which is a “stop” codon formost other prokaryotic and eukaryotic organisms. Hence any proteinscontaining tryptophan will not be expressed properly. Additionally, thepresent example is based on a whole unfractionated library. Thisincludes intergenic (non-coding regions). Moreover, only 1:6 of theinserts will be in the correct orientation to given expression. For allthese reasons it is clear that the vast majority of constructs in thelibrary will result in no protein (antigen) expression following “DNA ”vaccination.

Large-scale cultures of phage were grown on agar plates, harvested andthe phage particles purified and concentrated by a, combination ofprecipitation with polyethylene glycol and ultracentrifugation (SambrookJ, et al. 1989, Molecular cloning: a laboratory manual. Cold SpringHarbor Laboratory Press, N.Y.). Half of the phage suspension wasdirectly used to inoculate one group of mice (“Phage” DNA vaccine). DNAwas extracted from the other half of the phage suspension and used toinoculate a second group of mice (“Naked” DNA vaccine).

Sixteen mice were injected intramuscularly with 25 μl phage containingthe cloned F38 library at 4×10¹¹ particles/ml mixed with 25 μl montanideISA 206 adjuvant. A sample of this immunogen was titred and plated outon a lawn of E. coli to ensure that addition of montanide has not in anyway damaged the phage particles. A second set of sixteen mice weresimilarly injected with 0.5 μg of the extracted DNA (equivalent to theamount of DNA contained within the whole phage injection) in a totalvolume of 50 μl SM buffer. DNA was purified using Wizard lambda preps(Promega). A group of 8 mice were injected with purified C1857 phage inthe manner described for the phage library. This is also a lambda phageand was used as a negative control.

Mice were pre-bled before being inoculated on day 0. On day 34 anidentical booster injection was given. On day 72 the mice werechallenged with whole mycoplasma, before being exsanguinated on day 83.Test bleeds were taken every two weeks, until the conclusion of theexperiment. Bleeds were tested by ELISA against whole cells ofMycoplasma capricolum strains F38. In both the “Naked” DNA control andnegative phage (cI857) control groups, no significant response wasobserved until the final injection containing whole cells. By contrast,mice inoculated with the phage library showed a significant positiveresponse before the final injection was given (FIG. 1). In addition, therelative amount of the different subtypes of immunoglobulin present inthe final bleed was examined. It was found that in the mice vaccinatedwith the phage library the relative concentration of IgG to IgM wassignificantly higher (FIG. 2). High levels of IgG are indicative of asecondary response, whereas IgM is indicative of a primary response.

EXAMPLE 2

The uptake of phage particles directly by antigen presenting cells wasalso examined. In this instance the whole λ Mccp library was not used.Instead, a phage clone was picked which gave a good positive responseagainst polyclonal rabbit Mccp antiserum when plated on E. coli (proteinexpression was induced using IPTG). The clone was amplified andpurified. DNA was also extracted from this phage as previouslydescribed. Samples of DNA and whole phage particles of this clone wereadded to cultures of mouse pertitoneal macrophages. To extractmacrophage, 2 mice were injected intraperitoneally with 2 ml ofthioglycollate medium. 5 days later the mice were killed and theperitoneal cavity lavaged with 2 ml of magnesium/calcium free Hanksbuffered salt solution (HBSS) These extracted cells were pelleted andwashed twice in HBSS and a viable count performed in the presence oftrypan blue. 10⁶ macrophage were incubated at 37° C. in 1 ml RPMIcontaining 10% foetal bovine serum (FBS) for 2 h in 24 well microtitreplates to allow them to adhere. The supernatant containing non-adherentcells was removed and fresh medium added. 10⁹ phage particles of thisparticular clone were added to one well and 50 ng of DNA was extractedfrom this clone and added to fresh medium and cultured overnight at 37°C. Phage containing no insert and DNA extracted from these “no-insert”phage were used as negative controls.

After incubation the macrophage were placed at 4° C. to detach from themicrotitre plate, before they were harvested and washed in HBSS. 10⁵cells were then spun onto a glass slide and a cytostain performed usingrabbit Mccp polyclonal antiserum. The purified IgG fractioned was used.This antiserum was not further purified (ie. it was a polyvalentantiserum raised against whole Mccp, and thus recognises a broad rangeof Mccp proteins and other contaminants. As such, the signal to noiseratio is likely to be much lower than would be expected for a monovalentserum or a monoclonal antibody raised against the particular clone undertest). A secondary biotinylated antibody (goat anti rabbit wholeimmunoglobulin) along with an avidin/biotin/peroxidase DAB based systemwas used to visualise expressed protein.

Although high backgrounds were observed (probably due to the use of anon-specific polyclonal primary antibody, and the use of a secondaryantibody raised against whole rabbit immunoglobulin rather than the IgGfraction) more intense, specific staining was observed in the cellcultures spiked with insert-containing phage when compared to thenegative controls (FIG. 3).

EXAMPLE 3

A different expression library was tested, and modified vaccinationconditions were examined. A phage expression library was constructed bycloning semi-random fragments (Tsp509I): cuts at AATT) of the genome ofMycoplasma mycoides subspecies mycoides small colony biotype (MmmSC)strain T₁44 into the EcoRI site of the lambda ZAP expression vector. Thevector system, cloning and phage purification procedure followed weresimilar to those described in Example 1, although some differences inprocedure occurred during the vaccination phase (route ofadministration, presence of adjuvant, immunisation schedule). Ten miceper group were tested (BALB/C strain, specified pathogen free, age 10–12weeks). Mice were injected subcutaneously (without adjuvant) with 50 μlphage containing the MmmSC library at 1.7×10¹¹ particles per ml. Controlmice were not injected. Mice were pre-bled before being vaccinated onday 0. On day 28 an identical booster injection was given. Test bleedswere taken on Day 0, 21, and 42. Bleeds were tested by ELISA againstsonicated whole cell extracts of MmmSC strain T₁44. None out of 10control (non-immunised) mice showed an immune response. Two out of 10mice immunised with the whole MmmSC expression library showed asignificant immune response, while two others showed a lower levelresponse (FIG. 4).

EXAMPLE 4

A DNA construct expressing a specific antigen (green fluorescent protein[GFP]) was used to vaccinate mice and the immune response was measured.The vaccine antigen was presented to the immune system either aspurified plasmid DNA pEGFP-C1 (“naked” DNA) or as a bacteriophage GFPvaccine (λ-GFP). λ-GFP was constructed from the plasmid pEGFP-C1(Clonetech, catalogue no. 6084-1) and NM459 (obtained from NoreenMurray). NM459 is a cI⁸⁵⁷ nin derivative of phage construct XII from:Murray and Murray (1974). Manipulation and restriction targets in phageλ to form receptor chromosomes for DNA fragments. Nature, 251 p476–481.

pEGFP-C1 DNA was digested with EcoRI and the enzyme heat inactivatedbefore extraction with phenol: chloroform: isoamyl alcohol and ethanolprecipitation. NM459 DNA was also extracted and digested with EcoRI. Therestriction enzyme was then heat inactivated and the fragmentdephosphorylated (with calf intestinal alkaline phosphatase) to preventself-ligation during subsequent steps. This enzyme was then also heatinactivated and the DNA extracted with phenol: chloroform: isoamylalcohol and ethanol precipitated. These fragments were then ligated andthe cloned DNA packaged in vitro using a Promega Packagene Lambda DNApackaging system (Cat no. K3154).

After packaging 6 clones were picked from LB-agar plates. These wereamplified, DNA was extracted and digested with EcoRI to confirm thepresence of the insert. One of these clones was then amplified on alarger scale and concentrated stocks prepared for vaccination of mice.

Mice were strain C57, 8–10 weeks of age, 8 mice per group. Group 1 wasinjected intramuscularly on day 0 and day 14 with 1 μg of plasmid DNA(pEGFP-C1) in 0.1 ml of T.E. buffer. Group 2 were injectedintramuscularly on day 0 and day 14 with 50 μl of λ-GFP at 10¹¹particles per ml. Mice were bled on day 0 (prior to vaccination), day 14(prior to the second vaccination), and day 28. Bleeds were tested byimmunoblotting to detect a size-specific immune response againstrecombinant GFP, obtained from Clontech, Cat. No. 8360-2 (FIG. 5A) orbacteriophage λ proteins (FIG. 5B). Two out of 8 mice vaccinated withλ-GFP showed a strong immune response against GFP (at day 28 (FIG. 5A).Another mouse showed a response at day 14 which was lost by day 28 (datanot shown); it is possible that the day 14 and day 28 samples for thismouse became mixed up. Of the group vaccinated with plasmid pEGFP-C1,only 1 animal showed a response against GFP. The signal was much weakerthan the signal seen from the two positive animals which had beenvaccinated with λ-EGFP. All mice vaccinated with λ-EGFP showed a strongimmune response against bacteriophage λ proteins at days 14 and 28,while none of the mice vaccinated with plasmid pEGFP-C1 showed aresponse (FIG. 5B. Only bleeds from some animals are shown sinceidentical results were observed with all animals from both groups).

EXAMPLE 5

The uptake of phage particles directly by antigen presenting cells wasalso examined. Plasmid pEGFP-C1 was also tested as a control. For thisexperiment λ-GFP (described in Example 3) and another construct, λB1were tested. λB1 was isolated from the λ MmmSC expression librarydescribed in Example 3. λB1 contained the pyruvate dehydrogenase (pdh)gene of MmmSC (encoding a protein of 55 kD) and several otherunidentified open reading frames. When this clone was excised as aplasmid and transformed into Escherichia coli, two proteins wereidentified when these cell extracts were run out on SDS-PAGE gels,transferred to nitrocellulose and probed with rabbit hyperimmuneantiserum raised against MmmSC. The molecular weights of these twoproteins were 55 kD and 29 kD, and they were not observed using controlEscherichia coli extracts (containing the plasmid only, without the pdhinsert), or when Escherichia coli pdh-plasmid containing extracts wereprobed with control rabbit antiserum.

Samples of plasmid DNA and whole phage particles of these clones wereadded to cultures of mouse peritoneal macrophages. To extract themacrophages, 2 mice were injected intraperitoneally with 2 ml ofthioglycollate medium. Five days later the mice were killed and theperitoneal cavity lavaged with 2 ml of magnesium/calcium free Hanksbuffered salt solution (HBSS). These extracted cells were pelleted andwashed twice in HBSS and a viable count performed in the presence oftrypan blue. 10⁶ macrophage were incubated at 37° C. in 1 ml serum freeRPMI for 2 h in 24 well microtitre plates to allow them to adhere. Thesupernatant containing non-adherent cells was removed, the adherentcells were washed twice in PBS at 37° C., and 1 ml RPMI containing 10%foetal bovine serum (FBS) added. 10⁹ phage particles of clones λ-GFP andλ B1 were added to individual wells, or alternatively, 100 ng ofpurified plasmid pEGFP-C1 in T.E. buffer was added. The cells wereoverlaid with fresh medium and cultured overnight at 37° C.

After incubation the macrophage were placed at 4° C. for 1 h to allowthem to detach from the microtitre plate before they were harvested andwashed in HBSS. 5×10⁵ cells were then spun onto a glass slide and acytostain performed using either rabbit MmmSC polyclonal antiserum,1:1000 dilution in PBS (purified IgG fractioned), or rabbit GFPpolyclonal antiserum, 1:5000 dilution (obtained from Invitrogen, Cat.No. R970-01). Neither antiserum was affinity purified against GFP or theMmmSC pdh gene product, and therefore might be expected to give arelatively high background. A secondary biotinylated antibody (goat antirabbit whole immunoglobulin) along with an avidin/biotin/peroxidase DABbased system was used to visualise expressed protein.

Although some background signals were observed (probably due to the useof a non-specific polyclonal primary antibody, and the use of asecondary antibody raised against whole rabbit immunoglobulin ratherthan the IgG fraction), more intense, specific staining was observed inthe cell cultures spiked with insert-containing phage (GFP and B1) whencompared to the controls (FIG. 6A at 10× magnification, and FIG. 6B, 40×magnification). This staining was more noticeable with colour plates dueto the difference in signal between the specific brown staining of theperoxidase/DAB system seen with a positive signal, as compared to thegeneral violet staining of the macrophages. To confirm that staining wasnot due to antiserum binding to the bacteriophage lamba particles orpEGFP-C1 DNA that were added to the macrophages prior to visualisation,the following experiment was conducted. Phage particles (10⁹) orpEGFP-C1plasmid DNA (500 ng) were spotted onto a nitrocellulose filter,and incubated with the same primary and secondary antiserum used in theexperiments shown in FIGS. 6A and 6B. When developed using the sameprocedure, no staining was seen, indicating that any signal observed inthse figures could not be due to non-specific binding of the antiserumto either bacteriophage λ or plasmid DNA.

EXAMPLE 6

The uptake of phage particles and expression of vaccine antigen directlyby antigen presenting cells was also examined using a quantitative ELISAsystem. For this experiment λ-GFP (described in Examples 4 and 5) andplasmid pEGFP-C1 were tested. A standard non-expressing bacteriophage λconstruct was also tested as a negative control (λ-cI⁸⁵⁷). Macrophage(as described in Example 4) were removed from liquid nitrogen, washedtwice in serum/antibiotic-free RPMI medium, resuspended in the samemedium and 10⁶ cells per well added to the wells of a 96 well cultureplate. Macrophage were then incubated at 37° C. for 2 hours to allow thecells to attach, before the medium was removed and 100 μl fresh RPMI(containing 10% fetal calf serum+antibiotics) was added to each well.10⁹ phage (either λEGFP or negative control λ-cI⁸⁵⁷) or 50 ng pEGFP-C1DNA were added per test well. The test plate was then incubated at 37°C. overnight with 5% CO₂.

After incubation wells were washed four times in PBS, and antigenproduction was measured by ELISA using a Vector labs ABC biotin/avidindetection system (Cat. No. PK-6200). Primary antiserum was rabbitanti-GFP (Invitrogen). Bound secondary antibody was quantified usingSIGMA O-phenylenediamine dihydrochloride tablets (Cat. No. P-9187).Results were read in the culture plates at 492 nm and are shown in FIG.6. The mean ELISA value observed for macrophages given λ EGFP was2.03±0.25, significantly higher than that observed for macrophages givenpEGFP-C1 (mean value 1.54±0.08, p<0.05). The value observed withpEGFP-C1 was not obviously higher than that observed with the twonegative control assays, although since these were only performed induplicate a statistical comparison is not possible.

1. An immunogenic formulation consisting essentially of apharmaceutically acceptable carrier and a bacteriophage particlecomprising an exogenous nucleic acid under the control of a eukaryoticpromoter, wherein the exogenous nucleic acid encodes a polypeptide whichis capable of expression and presentation on the surface of an antigenpresenting cell of an organism such that an immune response to thepolypeptide is raised in the organism, said polypeptide is not designedto target the phage to receptors on the surface of specific cell types,and said bacteriophage has been modified to present said polypeptide onthe surface of the phage particle.
 2. The formulation according to claim1, wherein the polypeptide elicits an immune response against a virus,bacterium, fungus, yeast, protozoan, helminth, insect or transmissiblespongiform encephalopathy.
 3. The formulation according to claim 1,wherein the polypeptide elicits an immune response against a cancer cellspecific antigen.
 4. The formulation according to claim 1, wherein thebacteriophage comprises transcriptional and/or translational regulatorsto facilitate expression of the polypeptide in addition to a eukaryoticpromoter.
 5. The formulation according to claim 1, wherein theeukaryotic promoter is selected from the group consisting of a CMV,SV40, thymidine kinase and RSV promoter.
 6. The formulation according toclaim 1, wherein the eukaryotic promoter is a constitutive promoter. 7.The formulation according to claim 1, wherein the eukaryotic promoter isa controllable promoter.
 8. The formulation according to claim 1,wherein the bacteriophage is lambda (λ), pl phage, T phage, Mu, fd, M13or a filamentous phage.
 9. The formulation according to claim 1, whereinthe bacteriophage is abortive to lytic growth in the natural bacterialflora of the chosen mammalian host.
 10. The formulation according toclaim 1, further comprising inhibitors of lysosomal/endosomal enzymiccatabolism and/or nuclear localization signals.
 11. The formulationaccording to claim 1, further comprising an adjuvant.
 12. Theformulation according to claim 1, wherein the bacteriophage isassociated with a vehicle.
 13. An immunogenic formulation consistingessentially of (A) a pharmaceutically acceptable carrier, (B) abacteriophage particle comprising an exogenous nucleic acid under thecontrol of a eukaryotic promoter, wherein the exogenous nucleic acidencodes a polypeptide which is capable of expression and presentation onthe surface of an antigen presenting cell of an organism such that animmune response to the polypeptide is raised in the organism, and thesurface of the bacteriophage particle has not been modified to presentan exogenous peptide or protein designed to target the phage toreceptors on the surface of specific cell types, and (C) an amount ofthe polypeptide encoded by the exogenous nucleic acid.
 14. Animmunogenic formulation consisting essentially of a pharmaceuticallyacceptable carrier and a bacteriophage particle comprising an exogenousnucleic acid under the control of a eukaryotic promoter, wherein theexogenous nucleic acid encodes a polypeptide which is capable ofexpression and presentation on the surface of an antigen presenting cellof an organism such that an immune response to the polypeptide is raisedin the organism, the exogenous nucleic acid further encodes apolypeptide capable of augmenting the immune response, and the surfaceof the bacteriophage particle has not been modified to present anexogenous peptide or protein designed to target the phage to receptorson the surface of specific cell types.
 15. A method of treating diseasein a human or animal organism consisting essentially of administering animmunologically effective amount of a formulation consisting essentiallyof a pharmaceutically acceptable carrier and a bacteriophage particlecomprising an exogenous nucleic acid under the control of a eukaryoticpromoter, wherein the exogenous nucleic acid encodes a polypeptide whichis capable of expression and presentation on the surface of an antigenpresenting cell of the organism such that an immune response to thepolypeptide is raised in the organism, and the surface of thebacteriophage particle has not been modified to present an exogenouspeptide or protein designed to target the phage to receptors on thesurface of specific cell types.
 16. A method of treating diseaseaccording to claim 15, wherein the disease is an infectious disease. 17.A method of ameliorating disease in a human or animal organismconsisting essentially of administering an immunologically effectiveamount of a formulation consisting essentially of a pharmaceuticallyacceptable carrier and a bacteriophage particle comprising an exogenousnucleic acid under the control of a eukaryotic promoter, wherein theexogenous nucleic acid encodes a polypeptide which is capable ofexpression and presentation on the surface of an antigen presenting cellof the organism such that an immune response to the polypeptide israised in the organism, and the surface of the bacteriophage particlehas not been modified to present an exogenous peptide or proteindesigned to target the phage to receptors on the surface of specificcell types.
 18. A method of raising an immune response in a human oranimal organism consisting essentially of administering animmunologically effective amount of a formulation consisting essentiallyof a pharmaceutically acceptable carrier and a bacteriophage particlecomprising an exogenous nucleic acid under the control of a eukaryoticpromoter, wherein the exogenous nucleic acid encodes a polypeptide whichis capable of expression and presentation on the surface of an antigenpresenting cell of the organism such that an immune response to thepolypeptide is raised in the organism, and the surface of thebacteriophage particle has not been modified to present an exogenouspeptide or protein designed to target the phage to receptors on thesurface of specific cell types.